HIV and other enveloped RNA viruses bud from cells by usurping cellular machinery involved in multivesicular body (MVB) vesicle formation. To date, the ATPase, Vps4, is the only enzyme known to function in MVB vesicle formation, and it works together with LIP5/Vta1p to release/remodel the assembled MVB machinery. This remodeling activity allows multiple rounds of budding, and may also play an active role in vesicle/virus release. To gain important insights into HIV budding, I propose to investigate the molecular mechanism of Vps4/LIP5 function. I hypothesize that Vps4 releases the MVB machinery by pulling ESCRT-III protein substrates through the central pore of a homo-oligomeric ring structure, in analogy to Clp/Hsp100 bacterial charperones (which belong to the same superfamily of AAA ATPases). I propose a series of biochemical experiments that will test this idea by revealing whether: 1) VPS4 can be retargeted and shown to unfold heterologous GFP reporters, 2) substrates can bind to the center of the assembled Vps4 pore, and 3) ATP binds asymmetrically to a subset of the Vps4 subunits, allowing ATP hydrolysis to proceed in a "wave" around the ring and thereby drive ECRT-III translocation and unfolding. [unreadable] [unreadable] [unreadable]